Project Summary Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway whose function is to convert 5?-deoxy-5?-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work from our lab and others has established that MTAP can act as a tumor suppressor gene. However the precise mechanism by which MTAP loss promotes tumorigenesis is still unclear. One possible mechanism involves the accumulation of MTA, which is secreted by MTAP-deleted tumor cells. Structurally, MTA closely resembles adenosine and evidence indicates that it can interact with adenosine receptors. Large-scale genetic screens using shRNA have established that MTAP- deleted cells are especially sensitive to knockdown of a specific protein arginine methyltransferase enzyme (PRMT5), which is responsible for the post-translational symmetric dimethylation modification of arginine residues (sDMA). This modification is frequently observed in proteins involved in mRNA maturation. In preliminary data, we demonstrate that loss of MTAP or addition of extracellular MTA causes a dramatic reduction of the steady-state levels of sDMA-containing proteins. Significantly, when extracellular MTA is added, no increase in intracellular MTA occurs, suggesting that the reduction in sDMA-ylation is due to a signal transduction process. However, our data also suggests that enzymatically inactive MTAP protein itself, independent of MTA, can affect mRNA levels and antagonize the effects of MTA. Thus the overall goal of this proposal is to explore the hypothesis MTAP mediates its tumor suppressor function via two different mechanisms: one involving MTA as an oncometabolite and the other a direct role for the MTAP protein. The specific aims of the study are: (1) Identify specific proteins and arginine residues that are differentially methylated in response to MTAP; (2) Determine the roles of intracellular and extracellular MTA in the mechanism of sDMA-lyation in cancer cells; and (3) Clarification of the enzymatic vs. non-enzymatic functions of MTAP. These studies are significant because they provide a potential mechanism for understanding the role that MTAP deletion plays in tumorigenesis and may lead to novel therapeutic strategies for MTAP-deleted tumors. In addition, these studies will shed light on how a ?housekeeping? metabolic enzyme can have a novel role as a tumor suppressor gene.